Pure heptasulfated disaccharides having improved oral bioavailability

ABSTRACT

Hypersulfated disaccharides with utility in asthma or asthma related disorders are disclosed. The heptasulfated disaccharides administered orally have comparable bioavailability to the intravenous administered dosage form.

This application is a Divisional of U.S. application Ser. No. 16/325,940filed Feb. 15, 2019, which is a 371 of PCT Application No.PCT/US2017/046774 filed Aug. 14, 2017, which claims priority to U.S.Provisional Application No. 62/375,528 filed on Aug. 16, 2016, which areall incorporated herein to by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to substantially pure heptasulfateddisaccharide compounds of formula I and pharmaceutically acceptablesalts thereof and their use in the treatment of pulmonary disease andother inflammatory conditions. The purified compounds are particularlyuseful in the treatment of a variety of inflammatory disorders anddiseases in animals and people, and, in particular, pulmonary disordersselected from asthma and other conditions or diseases associated withinflammation of the lungs and airway.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 7,056,898 (the '898 patent) discloses and claims certainhypersulfated disaccharides and methods of using same to treat certaininflammatory disorders. This patent specifically describes the use ofthe compounds to treat pulmonary inflammation including asthma andasthma-related pathologies, such as allergic reactions or aninflammatory disease or condition. The compounds disclosed therein aredescribed as being capable of preventing, reversing and/or alleviatingthe symptoms of asthma and asthma-related pathologies, particularly thelate phase response in asthma patients following antigen stimulation.The examples and figures shown therein specifically relate tointravenous and inhalation means of administration of the reciteddisaccharides. In the '898 patent there is a general disclosure of theoral administration of a hexasulfated disaccharide designated as811-25-1 at a dose of 0.5 mgs/kg to sheep, but no specific data isshown. There is also no disclosure therein of any specific oralformulation nor any specific disclosure of any data related toadministration of a specific oral formulation. Additional patents havebeen published or granted that claim the compounds disclosed in the '898patent along with polymers that are used to enhance the lowbioavailability of the hexasulfated discaccharides disclosed in the '898patent-see U.S. Pat. No. 8,546,351. In fact, the delivery enhancingpolymers such as Carbopol® are described in the '351 patent as essentialin permitting the effective oral delivery of such molecules. Otherpublications and patent applications have disclosed other polysulfateddisaccharides including, among many other compounds, the molecule shownas formula I herein. See, for example, WO2006017727A which discloses acompound of formula (1):

wherein R1, R2, R3 and R4 are independently selected from H, C₁₋₄alkyl,—SO₃H, sulfated or unsulfated glycosyl or sulfated or unsulfateddiglycosyl group with the proviso that at least one of R1-R4 is sulfatedor unsulfated glycosyl or sulfated or unsulfated diglycosyl or saltsthereof. Specific examples are also shown and described therein but theprocesses described in this publication produce impure forms of theexamples. In addition, the processes disclosed require multipleprotecting groups at various steps and disclose processes that result ingreater than 1.5 wt % salt impurities in the final products. The presentinventors have discovered a novel process that produces substantiallypure crystalline polysulfated salts and, in particular, a substantiallypure form of 2, 5-Anhydro-3-O-(α-L-idopyranosyl)-D-mannitolhepta-O-sulfate salts. There is a need for heptasulfated disaccharidesproduced in substantially pure form and which are useful in highlybioavailable formulations for the treatment of pulmonary and otherinflammatory disorders.

There is no disclosure in WO2006017727A of the improved oralbioavailability or higher potency of any of the compounds or examplestherein nor is there any disclosure of the substantially pure forms ofthe drugs or of the particular dosage strengths recited and claimedherein for the treatment of asthma or any other indication. There is noteaching of oral dosage forms and strengths having improved LAR and EARin in vivo sheep models. Surprisingly and unexpectedly, the presentinventors have found that replacing the carboxylic acid moiety on thecompounds disclosed in the '898 and '351 patents with a sulfate group onsuch polysulfated compounds along with producing a substantially pureform leads to compounds having remarkable oral bioavailability. There isthus no need to improve the bioavailability of such compounds withpolymeric additives.

There is a need for an improved pulmonary or anti-inflammatorymedication that is both efficacious and can be delivered in dosages topatients in need of treatment thereof on a convenient basis and whichdoes not have the side effects associated with, for example, chronicadministration of steroids or leukotriene receptor antagonists such asmontelukast sodium. There is also a need to treat such diseases orconditions with a molecule having improved oral bioavailability in asuitable oral dosage form. In addition, there is a need for effectivedrugs that can be administered directly to the lungs in aerosol formthat include the recited heptasulfated disaccharides along with optionaland additional active pharmaceutical ingredients.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical dosage forms comprisinga substantially pure compound of formula I and pharmaceuticallyacceptable salts thereof and a pharmaceutical acceptable excipient. Thepreferred embodiment is salt of formula I wherein R₁-R₇ are SO3-Na+ Thecompounds in the dosage form are a compound of formula I or apharmaceutically acceptable salt thereof,

wherein R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are selected from the groupconsisting of SO₃H and pharmaceutically acceptable salts thereof. Thepresent invention also relates to dosage forms having a substantiallypure form of a compound of formula I. The invention further includespro-drugs, derivatives, active metabolites, partially ionized and fullyionized derivatives of the compounds of formula I and stereoisomersthereof. The monomers which make up the disaccharides of the inventionmay be D or L isomers and the hydroxyl moieties or sulfated versionsthereof around the carbocyclic ring (or acyclic versions orintermediates thereof) may have the alpha or beta designation at anyparticular stereocenter. The linking oxygen atom between themonosaccharide moieties may also be alpha or beta. The molecular weightof the compounds of the invention is typically less than 2,000 daltons.The preferred compound is 2,5-Anhydro-3-O-(α-L-idopyranase)-D-mannitolhepta-O-sulfate heptasodium salt.

The present invention also relates to a pharmaceutical dosage formcomprising

(i) a compound of formula I and pharmaceutically acceptable saltsthereof wherein R₁, R₂, R₃, R₄, R₅ and R₆ and R₇ are independentlyselected from SO₃H or PO₄H or salts thereof and(ii) a pharmaceutically acceptable excipient.

The present invention also encompasses a method of treating aninflammatory condition in an organism in need of treatment thereofcomprising administering a pharmaceutically effective amount of aformulation comprising a compound of formula I

and pharmaceutically acceptable salts thereof wherein R₁-R₇ areindependently selected from SO₃H or PO₄H.

The present invention also comprises a process for making asubstantially pure form of a compound of formula I comprising the stepsof preparing a compound of formula I wherein R₁₋₇ are H and sulfatingsaid compound of formula I with a sulfating reagent to form the compoundof formula I wherein R₁₋₇ are SO₃H or a salt thereof. In a preferredembodiment, the process comprises combining intermediate 1a withintermediate 1b to form a compound of formula I wherein R₁₋₇ is selectedfrom H.

In a more preferred embodiment, the present invention comprises aprocess for producing an intermediate compound of formula 1c, andprotected versions thereof, from intermediates 1a and 1 b and which isused to produce a compound of formula I herein in substantially pureform. In compound 1a, R is selected from a phenyl or tolyl moiety. Pr isa protecting group and may be selected from, for example, benzoyl orpivaloyl or other suitable protecting group. Thiophenyl or thiocresolare used as the thiolating reagents. Other thiolating reagents may alsobe used.

The invention further comprises producing a substantially pure form of acompound of formula I from compound Ic comprising the steps of sulfatingthe compound of formula Ic and then purifying the heptasulfateddisaccharide salt by running it through a Sephadex column; evaporatingthe solvent followed by trituration in ethanol. Other similar columnsand solvents may be used provided the salt is obtained in substantiallypure form.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the invention will be described in the following drawings.

FIG. 1A shows a graph comparing the percentage change in pulmonaryairflow resistance (measured as cm H₂O/L/sec) (i.e., the R_(L))following the indicated time after antigen administration (time=0) ofsheep's responses (n=5) to exposure to antigen only (closed circles)(control) and antigen plus a liquid oral dosage of 0.5 mg/kg×4 days (QD)of the heptasulfated disaccharide (sodium salt) designated asSSS-056-01*. The last dose was administered ninety minutes beforeantigen challenge (i.e., −1.5 hr). Data are shown as antigen-inducedmean plus or minus SE % change in R_(L) in sheep (n=5) exposed toantigen first with no drug and then again several weeks later withantigen plus SSS-056-01.

FIG. 1B shows a bar graph illustrating the effect of pretreatment onairway hyperresponsiveness (AHR) in allergic sheep. Data are shown asmean plus or minus SE PD₄₀₀ (airway responsiveness) in breath units atbaseline, 24 hours post-antigen challenge in a group of sheep (n=5)exposed to antigen first with no drug and then again with antigenseveral weeks later following pretreatment (90 minutes beforehand) withan oral dose of SSS-056-01 (0.5 mg/kg×4 days (QD)) in liquid form. PD₄₀₀is defined as the provocating dose of carbochol in breath units whichcaused a 400% increase in R_(L). One breath unit is one breath of 1%solution of carbochol. PD₄₀₀ is an indicator of airway responsiveness.The 0.5 mg/kg oral dose (QD for 4 days) inhibited EAR by 77%, LAR by 95%and AHR by 100%.

FIG. 2A shows a graph comparing the percentage change in pulmonaryairflow resistance (i.e., the R_(L)) following the indicated time afterantigen administration (time=0) of sheep's responses (n=5) to exposureto antigen only (closed circles) (control) and antigen plus an iv dose(0.5 mgs/kg×4 days (QD) of the heptasulfated disaccharide designated asSSS-056-01 (open circles). The last dose was administered 90 minutesbefore antigen challenge (i.e., 1.5 hours). Data are shown asantigen-induced mean plus or minus SE % change in R_(L) in sheep (n=5)exposed to antigen first with no drug and then again several weeks laterwith antigen plus SSS-056-01. The results show that there is nodifference in bioavailability between the intravenous administered doseand the oral dose shown in FIG. 1A administered once a day.

FIG. 2B shows a bar graph illustrating the effect of pretreatment onairway hyperresponsiveness (AHR) in allergic sheep. Data are shown asmean plus or minus SE PD₄₀₀ (airway responsiveness) in breath units atbaseline, 24 hours post-antigen challenge in a group of sheep (n=5)exposed to antigen first with no drug and then again with antigenseveral weeks later following pretreatment (1.5 hours) with an iv. Dose(0.5 mgs/kg×4 days (QD)) of SSS-056-01. The results again show thatthere is no difference between the iv route of administration(bioavailability) and the oral route shown in FIG. 1B. The 0.5 mg i.v.dose (QD for 4 days) inhibited EAR by 77%, LAR by 92% and AHR by 100%.

FIG. 3A shows a dose-response graph comparing the percentage change inpulmonary airflow resistance (measured as cm H₂O/L/sec) (i.e., theR_(L)) following the indicated time after antigen administration(time=0) of sheep's responses (n=5) to exposure to antigen only (closedcircles) (control) and antigen plus a liquid oral dosage at variousstrengths (0.25 mg/kg (open circles); 0.5 mg/kg (closed triangles) and 1mg/kg, (open triangles)) of the heptasulfated disaccharide designatedSSS-056-01 (open circles). Data are shown as antigen-induced mean plusor minus SE % change in R_(L) in sheep (n=5) exposed to antigen firstwith no drug and then again several weeks later with antigen after beingpretreated with various oral doses of SSS-056-01 administered 90 minbefore antigen challenge.

FIG. 3B shows a bar graph illustrating the effect of pretreatment onairway hyperresponsiveness (AHR) in allergic sheep. Data are shown asmean plus or minus SE PD₄₀₀ (airway responsiveness) in breath units atbaseline, 24 hours post-antigen challenge in a group of sheep (n=5)exposed to antigen first with no drug and then again with antigenseveral weeks later following pre-treatment with various oral doses ofSSS-056-01 (0.25 mgs/kg, 0.5 mgs/kg and 1.0 mgs/kg) administered 90 min.before the antigen challenge. +P<0.05 vs. baseline; and *P<0.05 vs.antigen control. The data in FIGS. 3A and 3B demonstrate that a singleoral dose of SSS-056-01 at 0.25 mg/kg was ineffective while 0.5 mg/kgand 1 mg/kg inhibited LAR (71% and 77% inhibition) and AHR (100%inhibition) without an effect on EAR.

FIG. 4A shows a graph comparing the percentage change in pulmonaryairflow resistance (measured as cm H₂O/L/sec) (i.e., the R_(L))following the indicated time after antigen administration (time=0) ofsheep's responses (n=8) to exposure to antigen only (closed circles,control) and antigen plus multiple (3× total; 1× every 12 hours) liquidoral doses of 0.25 mg/kg, 0.5 mg/kg and 1.0 mg/kg of the heptasulfateddisaccharide designated as SSS-056-01, three weeks apart (open circles,solid triangle and open triangle respectively). Data are shown asantigen-induced mean plus or minus SE % change in R_(L) in sheep (n=8)exposed to antigen first with no drug and then again several weeks laterwith antigen after being pretreated before antigen exposure with threedoses each of 0.25 mg/kg, 0.5 mg/kg and 1.0 mg/kg SSS-056-01 (1× each 12hr period, 3 weeks apart). Antigen challenge was ninety minutes afterthe last dose.

FIG. 4B shows a bar graph illustrating the effect of pretreatment onairway hyperresponsiveness (AHR) in allergic sheep. Data are shown asmean plus or minus SE PD₄₀₀ (airway responsiveness) in breath units atbaseline, 24 hours post-antigen challenge in a group of sheep (n=8)exposed to antigen first with no drug and then again with antigenseveral weeks later following pre-treatment before exposure with aliquid oral dose of SSS-056-01 (0.25 mg/kg, 0.5 mg/kg and 1.0 mgs/kg)administered 1×3 each 12 hours. Antigen challenge was ninety minutesafter the last mg/kg dose. The results show that the effect ofmulti-dose oral SSS-056-01 is cumulative. While 0.25 mg/kg×3 doses isineffective; 0.5 mgs/kg×3 and 1 mg/kg×3 inhibited EAR (30% and 54%inhibition), LAR (85% and 87% inhibition) and AHR (100% inhibition). 1mg/kg caused significantly greater inhibition of EAR than 0.5 mg/kgwhile the effect on LAR and AHR were comparable.

FIG. 5A shows the area under the curve for early phase(AUC-EAR_(0-4 hr)) from the data obtained in FIG. 4A.

FIG. 5B shows the area under the curve for the late phase(AUC-LAR_(4-8 hr)) from the data obtain in FIG. 4A.

FIG. 6A shows a graph comparing the percentage change in pulmonaryairflow resistance (measured as cm H₂O/L/sec) (i.e., the R_(L))following the indicated time after antigen administration (time=0) ofsheep's responses (n=5) to exposure to antigen only (closed circles)(control) and antigen plus a liquid oral dosage of 0.5 mg/kgadministered twice a day (BID) for a total of seven doses of theheptasulfated disaccharide designated as SSS-056-01 (open circles). Datashown are antigen-induced mean plus or minus SE % change in SR_(L) insheep (n=5) exposed to antigen first with no drug and then again severalweeks later with antigen after being pretreated before antigen exposurewith the seven doses, the last dose was administered 90 minutes beforethe antigen challenge.

FIG. 6B shows a bar graph illustrating the effect of pretreatment onairway hyperresponsiveness (AHR) in allergic sheep. Data are shown asmean plus or minus SE PD₄₀₀ (airway responsiveness) in breath units atbaseline, 24 hours post-antigen challenge in a group of sheep (n=5)exposed to antigen first with no drug and then again with antigenseveral weeks later following pretreatment with seven total dosesadministered every 12 hours before exposure with an oral dose ofSSS-056-01 (0.5 mg/kg). Antigen exposure occurred ninety minutes afterthe last 0.5 mg/kg treatment. The data in FIGS. 6A and 6B show that theeffect of multidose BID dosing of oral SSS-056-01 is cumulative. 0.5mg/kg oral doses (BID×7 doses) inhibited EAR by 76%, LAR by 96% and AHRby 100%. This is significantly better than 0.5 mg/kg×3 doses. BID dosing(×7) is comparable to QD dosing (×4).

FIG. 7A shows the effect of multi-dose oral SSS-056 on antigen-inducedEAR, LAR and AHR in sheep (QD dosing). Oral SSS-056-01 (0.5 mg/kg) wasadministered once daily in the morning×4 days, and antigen challenge wasperformed 90 minutes after the last dose (n=5). EAR and LAR are shown asantigen-induced % change in R_(L)±SE, without (control, closed circles)and after treatment with oral SSS-056-01 (open circles). The resultsalso show that the effect of multi-dose oral SSS-056-01, BID versus QDdosing is comparable.

FIG. 7B shows post-antigen AHR shown as mean±SE PD₄₀₀ for the baselineand 24 hours post-antigen without (control) and after treatment withSSS-056-01. +P<0.05 vs. Baseline; *P<0.05 vs. antigen control.

FIG. 8A shows the effect of single dose “inhaled” SSS-056 onantigen-induced EAR, LAR and AHR in sheep (n=5). Inhaled SSS-056-01 inbacteriostatic injection water was administered 30 minutes beforeantigen challenge. EAR and LAR are shown as antigen-induced % change inR_(L)±SE without (control) and after treatment with various doses ofSSS-056-01.

FIG. 8B shows post antigen AHR shown as mean+/−SE PD₄₀₀ for the baselineand 24 hour post-antigen, with (control) and after treatment with 5 mgand 10 mg inhaled SSS-056-01. This shows that 10 mg single dose ofinhaled SSS-056-01 inhibits LAR (75% inhibition) and AHR (100%inhibition) without an effect on EAR; while the 5 mg dose wasineffective.

FIG. 9A shows multi-dose inhaled SSS-056-01 (10 mg×3) had no significantcumulative effect on EAR (n=6), but did inhibit LAR. The data wascomparable to a single dose, as shown in FIG. 8.

FIG. 9B shows post antigen AHR shown as mean SE PD₄₀₀ for the baselineand 24 hour post-antigen, with (control) and after treatment with 10mg×3 inhaled SSS-056-01. This shows that multiple dose (10 mg×3) ofinhaled SSS-056-01 inhibits LAR (75% inhibition) and AHR (100%inhibition) without an effect on EAR.

FIG. 10 shows the proton NMR of SSS-056-01 (2,5-anhydro-3-O-(α-L-idopyranose)-D-mannitol-hepta-O-sulfate-hepta sodiumsalt).

*This compound is also described herein as SSS-02.

DETAILED DESCRIPTION

The present invention comprises a substantially pure form of a compoundof formula I:

wherein R₁, R₂, R₃, R₄, R₅ and R₆ and R₇ are independently selected fromthe group consisting of SO₃H or PO₃H or pharmaceutically acceptablesalts thereof.

The present invention relates to pharmaceutical formulations and usesthereof wherein the formulation comprises a substantially pure compoundof formula I and pharmaceutically acceptable salts thereof

wherein R₁, R₂, R₃, R₄, R₅ and R₆ and R₇ are independently selected fromthe group consisting of SO₃H or PO₃H.

The present invention also relates to a pharmaceutical formulationcomprising (i) a substantially pure form of a compound of formula I andpharmaceutically acceptable salts thereof

wherein R₁-R₇ are selected from SO₃H or pharmaceutically acceptablesalts thereof and (ii) a pharmaceutically acceptable excipient.

The present invention also relates to oral dosage forms comprising acompound of formula I and their pharmaceutically acceptable salts withR₁-R₇ as defined above.

The present invention further comprises a formulation comprising aproduct produced by a process of reacting compound 1a with compound 1bto form compound Ic and which is sulfated to form a substantially pureheptasulfated salt form of such compound and a pharmaceuticallyacceptable excipient.

The present invention also encompasses a method of treating oralleviating an inflammatory condition comprising administration of anoral dosage form of (i) a pharmaceutically effective amount of aformulation comprising a substantially pure compound of formula I

and pharmaceutically acceptable salts thereof wherein R₁-R₇ areindependently selected from SO₃H, PO₃H wherein the oral dosage form hascomparable bioavailability to an intravenous administered dosage form.

The present invention further relates to a method of treating aninflammatory condition comprising administering an oral dosage form ofbetween 0.25 to 10.0 mgs/kg/day to a patient in need of treatmentthereof.

The present invention also comprises i.v. formulations of thesubstantially purified form of a compound of formula I as well asinhalable formulations.

In a preferred embodiment, the compounds in the formulation are selectedfrom a metal salt of a compound of formula I wherein each sulfate orphosphate group around the disaccharide is ionized to form a metal saltwherein the metals are selected from, for example, sodium or potassium.In addition, other salts including amine salts may form at suchpositions. The most preferred compound is compound 1c in the fullysulfated and ionized form as the sodium salt.

It is generally understood that the source of the polysaccharide whichgenerates the disaccharides utilized in the formulations of theinvention will determine, for the most part, the absolutestereochemistry of the chiral centers around the carbohydrate rings.Additional sulfate groups are added by chemical means by the processdescribed generally above or by any known means to afford the mostactive moieties (hypersulfated disaccharides and salts thereof) whichare further purified to form pharmaceutical grade disaccharides whichare further formulated with an optional additive and processed into adosage form suitable for administration to a mammal or other organism inneed of treatment thereof.

Nuclear magnetic resonance imaging and/or other known structureidentification methods may be used to determine the chemical structuresof the molecules described herein (see FIG. 10).

A compound of formula I in the described strengths is formulated with apharmaceutically acceptable excipient to form the formulations of theinvention.

The formulations of the invention can be delivered to the patient orother organism by any suitable known means. The percentages of theadditive and type of additive added to the formulation relative to theactive ingredient and other excipients will be based upon the type offormulation desired. For example, in an oral suspension formulation tobe delivered to a patient or organism in need of treatment thereof, thevehicle can be an oral liquid or oral capsule. The preferred formulationis an oral capsule.

The compositions of the invention further comprise pharmaceuticallyacceptable excipients and/or fillers and extenders such as lactose orother sugars including but not limited to glucose, sucrose, mannitoletc. and lubricants such as magnesium stearate, talc, calcium stearate,solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof.The amount of filler or lubricant or other known pharmaceuticallyacceptable additive will vary based upon the type of formulation and themanner the formulation is processed or made.

The compositions of the invention can be delivered or administeredorally in the form of tablets, capsules or suspensions. The tablets orcapsules can be prepared by means known in the art and contain atherapeutically effective amount of a hypersulfated disaccharide offormula I with R₁₋₇ as defined herein. Tablets and pills or othersuitable formulations can be prepared with enteric coatings and otherrelease controlling coatings. Coatings can be added to afford lightprotection or swallowability. The capsules and tablets or suspensionscan include additives which improve the taste of the medicine.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixerscontaining inert diluents such as water as well as the compounds offormula I and salts thereof. Such formulations may additionally includeadjuvants including wetting agents, emulsifying and suspending agents,and sweetening, flavoring and perfuming agents. The compounds of theinvention may be formulated in a sustained release or delayed releaseformulation. The compounds may thus be formulated into extended releasedosage forms comprising a compound of formula I or a pharmaceuticallyacceptable salt thereof. Such formulations can include matrixformulations having extended release components such as cellulosicpolymers.

The compounds of formula I form, as stated above, pharmaceuticallyacceptable salts. The metal salts include for example salts having Na,K, Ca, Ng or Ba or Al, Zn, Cu, Zr, Ti, Bi, Mn or Os or salts formed byreacting the compounds of formula I with an organic base such as anamino acid or with any amine. The preferred salt is a sodium salt.

These formulations are useful in treating a number of inflammatorydiseases and conditions. Types of respiratory diseases or conditionscontemplated herein include allergic rhinitis which is characterized byseasonal or perennial sneezing, rhinorrhea, nasal congestion, and oftenconjunctivitis and pharyngitis; acute rhinitis, characterized by oedemaof the nasal mucosa, nasal discharge and mucosa. Pulmonary diseases,such as intrinsic or extrinsic asthma bronchiale, any inflammatory lungdisease, acute chronic bronchitis, pulmonary inflammatory reactionssecondary to chronic bronchitis, chronic obstructive lung disease,cystic fibrosis, pulmonary fibrosis, ARDS, acute lung injury,Goodpasture's syndrome as well as any lung disease or condition in whichwhite blood cells may play a role including idiopathic pulmonaryfibrosis and any other autoimmune lung disorders are treatable with theformulation of the invention.

Ear, nose and throat disorders such as acute external otitis,furunculosis and otomycosis of the external ear are treatable by theformulations of the invention. Other conditions include respiratorydiseases such as traumatic and infectious myringitis, acute Eustachiansalpingitis, acute serous otitis media and acute and chronic sinusitis.

Formulations of the invention are useful in treating pulmonaryinflammation. The term “pulmonary inflammation” encompasses anyinflammatory lung disease, acute chronic bronchitis, chronic obstructivelung disease, pulmonary fibrosis, Goodpasture's syndrome, and anypulmonary condition in which white blood cells may play a role includingbut not limited to idiopathic pulmonary fibrosis and any otherautoimmune lung disease.

Formulations of the invention are useful in treating asthma and asthmarelated pathologies. The term “asthma” means a condition of allergicorigin, the symptoms of which include continuous or paroxysmal laboredbreathing accompanied by wheezing, a sense of constriction in the chest,and, often, coughing or gasping. The term “asthma related pathologies”means a condition whose symptoms are predominantly inflammatory innature with associated bronchospasm. Both asthma and an asthma relatedpathology are characterized by symptoms which include a narrowing of theairways, varying over short periods of time either spontaneously or as aresult of a treatment, due in varying degrees to contraction (spasm) ofsmooth muscle, edema of the mucosa, and mucus in the lumen of thebronchi and bronchioles. Generally these symptoms are triggered by localrelease of spasmogens and vasoconstrictive substances (e.g. histamine orcertain leukotrienes or prostaglandins) in the course of an allergicresponse. Non-limiting examples of asthma related pathologies includenon-asthmatic conditions characterized by airway hyperresponsiveness(e.g. chronic bronchitis, emphysema and cystic fibrosis). The mostprominent characteristic of asthma is bronchospasm, or narrowing of theairways: asthmatic patients have prominent contraction of smooth musclesof large and small airways, increased mucous production, and increasedinflammation. The inflammatory response in asthma is typical for tissuescovered by mucosa and is characterized by vasodilation, plasmaexudation, recruitment of inflammatory cells such as neutrophils,monocytes, macrophages, lymphocytes, and eosinophils to the sites ofinflammation and the release of inflammatory mediators by residenttissue cells (mast cells) or by migrating inflammatory cells (J. C.Hogg, “Pathology of Asthma,” Asthma and Inflammatory Disease, P. O'Byren(ed.), Marcel Dekker, Inc., New York, N.Y. 1990, pp. 1-13).

Asthma may be triggered by multiple or a variety of causes such as inresponse to allergens, secondary exposure to infective agents,industrial or occupational exposures, ingestion of chemicals, exerciseand/or vasculitis (Hargreave et al., J. Allergy Clinical Immunol.83:1013-1026, 1986). As discussed herein, there may be two phases to anallergic asthma attack-an early phase and a late phase which follows 4-6hours after bronchial stimulation (Harrison's Principles of InternalMedicine 14^(th) Edl, Fauci et al. (eds), McGraw Hill, New York, N.Y.1998, pp. 1419-1426). The early phase which typically resolvesspontaneously, includes the immediate inflammatory response includingthe response caused by the release of cellular mediators from mastcells. The late phase reactions develop over a period of hours and arecharacterized histologically by an early influx of polymorphomuclearleukocytes and fibrin deposits followed by infiltration of eosinophils.A certain percentage of patients are “dual responders” and develop anearly acute and a late phase response. In dual responders, the acutephase is followed 4-14 hours later by a secondary increase in airwayresistance (“late phase response” or LPR or “late airway response” orLAR). Late responders and dual responders are of particular clinicalimportance because, in combination with the airway inflammation, latephase responses lead to a prolonged airway hyperreactivity (AHR),asthmatic exacerbations, or hyperresponsiveness, worsening of symptoms,and generally a more severe form of clinical asthma that may last fromdays to months in some subjects, requiring aggressive therapy.Pharmacological studies in allergic animals have demonstrated that notonly the bronchoconstrictor response but also the inflammatory cellinflux and the mediator release pattern in dual responders is quitedifferent from acute responders.

An increase in bronchial hyperreactivity (AHR), the hallmark of a moresevere form of asthma, can be induced by both antigenic andnon-antigenic stimuli. Late phase response, allergen-induced asthma andpersistent hyperresponsiveness have been associated with the recruitmentof leukocytes, and particularly, eosinophils, to inflamed lung tissue(W. M. Abraham et al., Am. Rev. Respir. Dis. 138: 1565-1567, 1988).Eosinophils release several inflammatory mediators including 15-HETE,leukotriene C4, PAF, cationic proteins and eosinophil peroxidase.

Moreover, the formulations of the invention are also useful in treatinglate phase reactions and inflammatory response in extra pulmonary sitessuch as allergic dermatitis, inflammatory bowel disease; rheumatoidarthritis and other collagen vascular diseases, glomerulonephritis,inflammatory skin diseases and conditions; and sarcoidosis.

As used herein, the term “treating or alleviating the symptoms” meansreducing, preventing and/or reversing the symptoms of the individual towhich a formulation of the invention has been administered as comparedto the symptoms of the individual or an individual which is untreated.Hence, a formulation of the invention that treats or alleviates thesymptoms of asthma or an asthma related pathology reduces, prevents,and/or reverses the early phase asthmatic response to antigen challengein a dual responder individual, more preferably reduces, prevents and/orreverses the late phase asthmatic response to antigen challenge in adual responder individual, and more preferably reduces, prevents and/orreverses both the early phase and late phase responses to antigenchallenge in a dual responder individual. This “treatment” or“alleviation” is preferably a significant percentage as shown in theanimal models presented herein for the recited formulations and withrespect to LAR and AHR data.

The terms “antigen” and “allergen” are used interchangeably to describethose substances such as dust or pollen that can induce an allergicreaction and/or induce an asthmatic episode or asthmatic symptoms in anindividual suffering from such condition. Thus an individual is“challenged” when an allergen or antigen is present in a sufficientamount to trigger an asthmatic response in such individual.

The term “substantially pure” means that the purified compound has lessthan 1.5 weight percentage of any impurity exclusive of solvents ormoisture.

It is also understood that the formulations of the invention are usefulin treating any disease or condition affected by late phase reactions(LPR's). The airways are merely a prototype of organs or tissuesaffected by such LPR's. It has been established in the medicalliterature that the last phase bronchoconstriction and AHR observed indual responder asthmatic patients is not an isolated phenomenonrestricted to asthmatic or even pulmonary patients. Thus, the presentformulation is useful in treating any disease or condition affected byLPR's including cutaneous, nasal, ocular and systemic manifestations ofLPR's in addition to pulmonary associated LPR's. Clinical diseases(whether of the skin, lung, nose, eye or other organs) recognized toinvolve allergic mechanisms have a histologic inflammatory componentwhich follows the immediate allergic or hypersensitivity reaction thatoccurs on antigen challenge. This sequence of response appears to beconnected to mast cell mediators and propagated by other resident cellswithin target organs or by cells recruited into the sites of mast cellor basophilic degranulation. Thus, the present formulation is useful intreating inflammatory bowel disease, rheumatoid arthritis,glomerulonephritis and inflammatory skin disease. The present inventiontherefore relates to a method of treating a patient or organism in needof treatment thereof and who/which is suffering from a disease orcondition characterized by late phase allergic reactions, including e.g,and without limitation, pulmonary, nasal, cutaneous, ocular and systemicLPR's, and/or which is characterized by inflammatory reactions throughthe administration, by any known means, of a formulation comprising acompound of formula I or II and a delivery agent such as, for example, apolymeric additive to said patient or organism.

The term “inflammatory condition” means a disease, condition or symptomselected from the group consisting of pulmonary inflammation such asasthma and/or asthma related pathologies; pneumonia, tuberculosis,rheumatoid arthritis, allergic reactions which impact the pulmonarysystem, early and late phase responses in asthma and asthma relatedpathologies, diseases of the small and large airways of the lung,bronchospasm, inflammation, increased mucus production, conditions whichinvolve vasodilation, plasma exudation, recruitment of inflammatorycells such as neutrophils, monocytes, macrophages, lymphocytes andeosinophils and/or release of inflammatory mediators by resident tissuecells (mast cells); conditions or symptoms which are caused byallergens, secondary responses to infections, industrial or occupationalexposures, ingestion of certain chemicals or foods, drugs, exercise orvasculitis; conditions or symptoms which involve acute airwayinflammation, prolonged airway hyperreactivity, increases in bronchialhyperreactivity, asthmatic exacerbations, hyperresponsiveness;conditions or symptoms which involve the release of inflammatorymediators such as 15-HETE, leukotriene C4, PAF, cationic proteins oreosinophil peroxidases; conditions or symptoms which relate tocutaneous, nasal, ocular or systemic manifestations of late phaseallergic responses; clinical diseases of the skin, lung, nose, eye orthroat or other organs and which involve allergic mechanisms having anhistologic inflammatory component upon antigen challenge; allergicrhinitis, respiratory diseases characterized by seasonal or perennialsneezing; rhinorrhea, conjunctivitis, pharyngitis, intrinsic orextrinsic asthma bronchiale, any inflammatory lung disease, acutechronic bronchitis, pulmonary inflammatory reactions secondary to acutechronic bronchitis, chronic obstructive lung disease (COPD), pulmonaryfibrosis, Goodpasture's syndrome, any pulmonary condition in which whiteblood cells play a role including but not limited to idiopathicpulmonary fibrosis and any other autoimmune lung disease; ear, nose andthroat disorders such as acute external otitis, furunculosis andotomycosis of the external ear; respiratory diseases such as traumaticand infectious myringitis, acute eustachian salpingitis, acute serousotitis media, acute and chronic sinitis; extrapulmonary conditionsselected from any late-phase reactions and inflammatory response such asallergic rhinitis; allergic dermatitis; allergic conjunctivitis;extrapulmonary diseases where inflammation occurs and/or an inflammatoryresponse plays a major role including inflammatory bowel disease;rheumatoid arthritis and other collagen vascular diseases;glomerulonephritis; inflammatory skin diseases and sarcoidosis andcardiovascular inflammation as described below. The compounds may alsobe used to treat central nervous system disorders such as Alzheimer'sdisease, including the inhibition of β-amyloid aggregation inAlzheimer's disease and neuroinflammation.

The present substantially pure compounds and formulations thereof mayalso be utilized to treat inflammatory conditions associated withcardiovascular disease. It is known that there are serious side effectsassociated with traditional anti-inflammatory agents such asglucocorticoid steroids and cyclophosphamide making them inappropriatechoices for atherosclerotic inflammation treatment. On the other hand,the polysulfated disaccharide formulations of the invention have theadvantage of having few side effects along with anti-inflammatoryproperties. It has clearly been postulated that atherosclerotic lesionsare due to or have many properties associated with chronic inflammationincluding the presence of macrophages, lymphocytes and denditric cellswhich accumulate at specific loci to cause and/or acerbate lesions. L.K. Curtiss, N. Engl. J. Med. 360; 11 1144-1146 (2009). The presentformulation is thus useful for the treatment of arterioscleroticdisorders in patients having such disorders or conditions and is furtheruseful in the treatment or prevention of restenosis after invasivevascular surgery or after an organ transplant. The formulation suitablefor cardiovascular treatment can be administered by any known meansincluding by interal or parenteral administration. The present inventioncomprises a method of treating cardiovascular inflammation comprisingadministration of a composition comprising a substantially pure compoundof formula I wherein R1-R7 are as defined herein and pharmaceuticallyacceptable salts thereof and a pharmaceutically acceptable excipient toa patient in need of treatment thereof. The present invention furtherincludes combinations of a compound of formula I with R1-R7 as definedherein and a cardiovascular drug selected from an HMGCoA reductaseinhibitor or other cardiovascular drug or drugs used to treatcardiovascular disease. The “combination” may be in the form of a singledosage form having at least two active ingredients wherein one of theactive ingredients is a hypersulfated disaccharide of the invention andthe other active ingredient is selected from an HMGCoA reductaseinhibitor such as lovastatin, simvastatin, atorvastatin or rosavastatincalcium. In a preferred embodiment, the combination would include aformulation of the invention comprising a compound of formula I or IIwherein R₁-R₇ is as defined herein and a second active ingredientselected from an HMGCoA reductase inhibitor.

The formulations of the invention have been found to be effective inanimal studies which are predictive of utility in humans as well asother animals. The animal studies demonstrate that the substantiallypure compounds and formulations thereof are useful in (a) preventingantigen-induced bronchoconstrictor response and bronchial hyperactivity(also referred to as airway-hyperresponsiveness (AHR)) and (b) inameliorating AHR subsequent to antigen challenge in treated animals.Pulmonary airflow resistance was measured by taking allergic sheeppreviously verified as dual bronchoconstrictor responders to Ascarissuum antigen. The sheep were intubated with a cuffed nasotracheal tubeand pulmonary airflow resistance (R_(L)) was measured by the esophagealballoon catheter technique. Airway responsiveness was determined byfirst securing cumulative dose response curves to inhaled carbachol (aconstrictor agonist) by measuring R_(L) before and after inhalation ofbuffered saline and after each administration of 10 breaths ofincreasing concentrations of carbachol (0.25, 0.5, 1.0, 2.0 and 4.0%wt/vol solution). Airway responsiveness was measured by determining thecumulative provocation dose (PD₄₀₀) of carbachol (in breath units) thatincreased R_(L) to 400% above baseline. One breath unit was defined asone breath of 1% carbachol solution.

As appropriate, and according to the prescribed method ofadministration, the substantially pure compounds of formula I as definedherein and formulations thereof may be administered prior to, at thesame time, or after the organism or patient has been exposed to anantigen and in relation to the particular disease or condition beingtreated. Doses of the active ingredient (the hypersulfated disaccharidesof formula I) preferably range from 1 mgs/kg to 5 mg/kg per day. Thesedoses may be adjusted by the physician depending upon the response ofthe patient. Oral doses are preferred. Suitable dose ranges may include0.05 mgs/kg/day to 25 mgs/kg/day.

The formulations of the invention may be administered alone or incombination with other suitable medications or active ingredients anddepending upon the particular disease or condition being treated. In apreferred embodiment, the formulations or compounds of the invention areadministered in the morning or evening. Thus, the present inventioncomprises a method of treating a disease or condition associated withantigen exposure and which involves an early and late phase responsecomprising administering to an organism in need thereof atherapeutically effective amount of a compound of formula I with R₁-R₇as defined herein wherein the formulation is administered in the morningor evening. The invention further comprises a method of treating adisease or condition associated with antigen exposure and which involvesan early and late phase response comprising administering to an organismin need thereof a therapeutically effective amount of a compound offormula I with R₁-R₇ as defined herein to form a formulation and whereinsaid formulation is administered to the organism in the morning orevening. The additional active ingredients that may be administered inthe form of combination therapy or in the form of a single dosage unithaving at least two active ingredients wherein the first active is acompound of formula I with R₁-R₇ as defined herein and a second activeselected from any drug or medicament which is used as front line therapyto treat asthma or an asthma related disorder or condition or otherinflammatory condition as recited herein. Such medicaments includeanti-inflammatories, leukotriene antagonists or modifiers,anticholinergic drugs, mast cell stabilizers, corticosteroids,immunomodulators, beta-adrenergic agonists (short acting and longacting), methyl xanthines, and other general classes or specific drugsused to treat such disorders including, but not limited to, montelukastsodium; albuterol; levoalbuterol; salmeterol; vilanterol, indacaterol,formoterol, fluticasone propionate; budesonide; ceterizine; loratadine;desloratadine; theophylline, ipratropium, cromolyn, nedocromil,beclomethasone, flunisolide, mometasone, triaminoclone, prednisoline,prednisone, zafirlukast, zileuton or omalziunab and other disaccharides.

The substantially pure compounds of the invention have surprisingly highbioavailability relative to, for example, hexasulfate disaccharides orrelative to impure forms of the drug. It is postulated that even thepresence of minor salt impurities (e.g. 1.5-2%) renders the drug lessbioavailable. The combination of both purity and the elimination of thecarboxylic acid group on hexasulfated analogs also yields surprisinglyhigh relative bioavailability. The present inventors have found that anoral dosage form of the substantially pure form of 2,5-anhydro-3-O-(α-L-idopyranosyl)-D-mannitol hepta-O-sulfate sodium salthas comparable bioavailability to the intravenous form. In addition, theinventors have found that the heptasaccharide is more potent on a mg/mgbasis than the same dosage strength of the comparable hexadissachridehaving a carboxylic acid moiety or salt thereof and, furthermore, doesnot require a polymeric additive to improve bioavailability.

The compounds of formula I were prepared by a process which synthesizedtwo building blocks-an α-idopyranose and a D-mannitol. In general, asshown in the scheme below, the two building blocks are reacted with eachother to form the disaccharide which is further processed to form thepolysulfated salts. The present inventors have found deficiencies in theprior art processes that led to significant impurities in the finalproduct. The inventors have solved the problems of creating a purerproduct by directly purifying the final product on a Sephadex column,followed by evaporation of the solvent and trituration with ethanol.This process avoids contamination by traces of sodium acetate which arein the final product produced by the process disclosed in WO20060177727using other solvents such as methanol and different purificationprocedures. Surprisingly, the replacement of methanol with ethanolyielded significant and unpredictable improvements. In addition to thisimprovement, the inventors also found that the prior art processesproduced low yields and were difficult to follow and produce large scalebatches. Furthermore, such processes required two different protectinggroups in the synthesis of the thioglycoside which then requiresmultiple deprotection steps. In the present process, the inventors havefound that use of the bridged idopyranosyl in crystalline form and witha single protecting group (Bz) is scaled up with ease and does notrequire the use of multiple protecting groups which require subsequentremoval. Also, in the coupling reaction of the present invention, theintermediates were solid and thus avoided the requirement of columnpurification and could be readily purified by crystallization methods.

The following examples are intended to further illustrate certainembodiments of the invention and are non-limiting.

Example 1-Preparation of Hypersulfated Disaccharides Having C-1 Sulfated

The compounds of the invention were prepared by the following generalscheme:

Preparation of Thiophenyl-L-idose Unit (First Block)

Literature: 1. J. Carbohydrate Chemistry, 1985, 4(3), 293-321

2. Carbohydrate Research, 2008, 343, 596-606

Preparation ofPhenyl-6-O-acetyl-2,3,4-tri-O-benzoyl-1-thio-α-L-idopyranoside

Modified and Optimized Procedure

Diacetone-D-glucose (2.0 mol, 520.0 g) and KI (0.2 mol, 33.0 g) weredissolved in dry DMSO (1500 ml) under argon and NaOH (1.15 eq, 2.3 mol,92.0 g) was added in one portion. After 15 min of stirring at 25° C.(almost complete dissolution), Benzyl chloride (1.05 eq, 2.1 mol, 266 g,242 ml) was slowly added keeping reaction temperature in the range25-35° C. Then, the reaction was stirred at 25° C. for additional 2 h(HPLC/TLC monitoring; Hep/EtOAc 1:2), poured into the mixture of Water(5 L) and MTBE (4 L) and the resulted mixture was stirred for 30 min.The phases were separated, the aqueous one was extracted with MTBE(2×700 ml) and the combined organics were washed with Water (2×800 ml)and brine (800 ml), dried over Na₂SO₄, filtered and evaporated underreduced pressure.

The residue (740 g) was dissolved in MeOH (3 L), a solution was cooledto 10° C. and treated with 1% aqueous H₂SO₄ (1 L). The reaction wasstirred at 30° C. for 48-72 h (TLC control; Hep/EtOAc 1:2) and quenchedby addition of solid NaHCO₃ (40 g). Slow gas evolution was observed.After 1 h of progressive stirring all volatiles were removed underreduced pressure and the syrupy residue was portioned between EtOAc (5L) and Water (3 L). The phases were separated, the aqueous one wasextracted with EtOAc (2×1 L) and the combined organics were washed withbrine (1 L), dried over Na₂SO₄, filtered and evaporated under reducedpressure. The syrupy residue (650 g) which contained 90-95% of thedesired 1,2-diol, contaminated with 2-5% of tetra-ol, was used withoutadditional purification.

The crude (650 g) and dry Pyridine (6 eq, 12 mol, 970 ml) were dissolvedin dry CH₂Cl₂ (3 L) and the solution was cooled to −25° C. A solution ofBenzoyl chloride (Note 1; 1 eq, 2.0 mol, 233 ml) in dry CH₂Cl₂ (500 ml)was slowly added at the rate which kept the reaction temperature below−15° C. (˜30 min period) and the resulted mixture was stirred foradditional 2 h at −20° C. (TLC control; CH₂Cl₂/EtOAc 10:1). Then,Methanesulfonyl chloride (1.5 eq, 3.0 mol, 233 ml) was slowly addedfollowed by addition of DMAP (0.2 eq, 0.4 mol, 49 g) and the reactionwas heated to 20° C. and stirred for additional 12 h (TLC control). Thereaction was quenched by addition of ice-water (3 L) and two-phasemixture was vigorously stirred for 30 min. The phases were separated,the aqueous one was extracted with CH₂Cl₂ (2 L) and the combinedorganics were washed subsequently with cold 2N aqueous HCl (1 L), water(500 ml) and 10% aqueous NaHCO₃ (500 ml), dried over Na₂SO₄, filteredand evaporated under reduced pressure. The semisolid residue (˜1 kg) wastriturated with MeOH (4 L) at 50° C. for 1 h. The resulted suspensionwas then slowly cooled to 0° C., stirred for 30 min and filtered. Thecake was washed with cold (2-8° C.) MeOH (2×500 ml) and Heptane (1 L),and dried under reduced pressure to give 837 g (85% yield) of Bz-Msderivative as white solid.

Note 1:

Pivaloyl chloride (l eq, 2.0 mol, 246 ml) may be used in the same mannerinstead of Benzoyl chloride to give about 80% of crude Piv-Ms derivativeas semisolid

A solution of Bz-Ms (507 g) in dry CH₂Cl₂ (3500 ml) and t-Butanol (1000ml) was cooled to 0° C. under Argon and Potassium t-butoxide was added(247 g, 2.2 mol) by portions. The resulted mixture was stirred for 5 hat 0° C., then for 7 h at 20° C. (TLC monitoring, Heptane/EtOAc 1:1) andconcentrated under reduced pressure. The residue was portioned betweenMTBE (2500 ml) and ice-cold water (1 L), the pH was adjusted to 7 byaddition of acetic acid and the mixture was filtered through Celite. Thephases were separated, the aqueous one was extracted with MTBE (1000 ml)and the combined extracts were washed with water and brine, dried overNa₂SO₄, filtered and concentrated under reduced pressure to give 450 gof brown oil. The residue was purified on a Silica gel (1 kg,Heptane/Ethyl acetate 10:1-4:1) to give 250 g of the desired5,6-anhydro-L-idofuranose as yellow oil with ˜90% purity by HPLC.

A solution of 3-O-Benzyl-5,6-anhydro-L-idofuranose (250 g) in 2M aqueousH₂SO₄ (450 ml) and 1,4-Dioxane (450 ml) was stirred under reflux for 12h. The reaction was cooled to 10° C. and neutralized with 10N aqueousNaOH. The most of Dioxane was evaporated under reduced pressure, morewater (1 L) was added and the aqueous mixture was extracted with EtOAc(3×800 ml). The combined organics were washed with Water and brine,dried over Na₂SO₄, filtered and concentrated under reduced pressure togive ˜200 g of semisolid residue. The residue was crystallized from EtOH(700 ml) afforded 150 g (59% from diacetone-D-glucose) of1,6-anhydro-3-O-benzyl-β-L-idopyranose as white solid, mp 157-158° C.(lit. 158-159° C.; JACS, 2001, 123, 3153-3154). The filtrate wasconcentrated to a half volume and refrigerated for 12 h to give secondcrop (20 g, 8%) of 1,6-anhydro-3-O-benzyl-β-L-idopyranose.

1,6-anhydro-3-O-benzyl-β-L-idopyranose (150 g, 0.6 mol) was dissolved inthe mixture EtOAc/MeOH (1:1, 1200 ml) and hydrogenated over 10% Pd/C (15g) at 50° C., 5 atm for 4 h. The mixture was cooled to 20° C., filteredthrough Celite and evaporated under reduced pressure. The oily residueand DMAP (22 g, 0.18 mol) were dissolved in CH₂Cl₂ (400 ml) and Pyridine(404 ml, 5.0 mol) and the resulted mixture was cooled to 0° C. Benzoylchloride (276 g, 229 ml, 1.96 mol) was slowly added and the reaction wasstirred for 3 h at 20° C. (TLC monitoring). The volatiles wereevaporated under reduced pressure and the solid residue was portionedbetween EtOAc (2 L) and water (2 L). The phases were separated, theorganic one was washed with cold 2N aqueous HCl (700 ml), water (500 ml)and 10% aqueous NaHCO₃ (500 ml), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give ˜300 g of solid residue. Theresidue was crystallized from Isopropanol afforded 265 g (94% yield) ofthe desired tribenzoate as white solid.

A solution of Tribenzoate (265 g, 0.56 mol) in CH₂Cl₂ (1000 ml) andAcetic anhydride (1000 ml, 10.5 mol) was cooled to 0° C. and TMSOTf (10ml, 51.5 mmol) was slowly added. The reaction was stirred for 1 h at 0°C. (TLC monitoring), quenched by addition of Et₃N (25 ml) andconcentrated under reduced pressure. The solid residue was re-evaporatedtwice with Toluene and crystallized from Isopropanol (1200 ml) afforded324.5 g (95% yield) of desired di-O-acetyl as white solid.

3) A solution of Di-O-acetyl (324.5 g, 0.56 mol) and Thiophenol (80.5ml, 0.79 mol, 1.4 eq), Note 2, in CH₂Cl₂ (2 L) was cooled to 0° C. andBF₃.Et₂O (89 ml, 0.70 mol, 1.25 eq) was slowly added. The reaction wasstirred for 12 h at 20° C. (TLC monitoring) and slowly poured (gasevolution!) into 10% aqueous NaHCO₃ (1500 ml). After 30 min of stirring,the layers were separated; the organic one was washed with water (I L),dried over Na₂SO₄, filtered and evaporated under reduced pressure. Thesolid residue (˜350 g) was crystallized from Isopropanol afforded 300 g(85% yield) ofPhenyl-6-O-acetyl-2,3,4-tri-O-benzoyl-1-thio-α-L-idopyranoside as whitesolid.

Note 2:

Thiocresol may be used instead of thiophenol afforded 87% ofTolyl-6-O-acetyl-2,3,4-tri-O-benzoyl-1-thio-α-L-idopyranoside as whitesolid

Preparation of 1,6-di-O-pivaloyl-2,5-anhydro-D-mannitol (Second Block)

The literature known procedure was modified and applied

(Tetrahedron: Assymetry 2000, 11, 2899-2906)

D-Glucosamine hydrochloride (400 g, 1.85 mol) was stirred in water (3000ml) for 24 h at 25° C. to reach mutarotational equilibrium. Then, thesolution was cooled to 0° C. and NaNO₂ (383 g, 5.6 mol, 3 eq) was addedin several portion. While the stirring and keeping the temperature below4° C. (exothermic reaction!), concentrated (37%) hydrochloric acid (315ml, 3.7 mol) was added dropwise to form nitrous acid in situ (stronglyexothermic reaction!). After additional 5 h of stirring at 0° C., thereaction was heated to 25° C. under flow of Argon in order to removeexcess nitrous acid and then it was neutralized to pH 7 with 10N aqueousNaOH. The resulted solution was cooled to 0° C. and NaBH₄ (70.0 g, 1.85mol) was added in small portions (gas evolution!). After 12 h ofstirring at 25° C., the reaction mixture was carefully neutralized with6N aqueous HCl and then concentrated under reduced pressure. Theremaining semisolid material was treated twice with MeOH at 50° C. andthe combined methanolic extracts were then concentrated under reducedpressure. The residue was extracted with Isopropanol (3×1000 ml) and thecombined extracts, Note 1, were evaporated to dryness under reducedpressure to give 225 g of the crude 2,5-anhydro-D-mannitol as a yellowsyrup, Note 2.

Notes

1: To remove all inorganic impurities, isopropanolic solution may bepassed through ion-exchange resin columns (anionic and cationic forms)2: According to literature, 2,5-anhydro-D-mannitol is solid and may becrystallized from EtOH or Isopropanol with seeding.

A mixture of 2,5-anhydro-D-mannitol (225 g, 1.37 mol) in dry pyridine(1000 ml) and dichloromethane (1000 ml) was cooled to −15° C. andBenzoyl chloride (366 g, 302 ml, 2.6 mol, 1.9 eq), Note 3, was addeddropwise. The reaction was stirred for 1 h at −10° C., 4 h at 0° C. andthen 10 h at ambient temperature (TLC monitoring in EtOAc). Allvolatiles were evaporated under reduced pressure and the residue wasportioned between ice-cold 3N aqueous HCl (1000 ml) and EtOAc (3000 ml).The phases were separated; the organic one was washed with water (500ml) and brine (300 ml), dried over Na₂SO₄, filtered and evaporated. Theresidue (˜550 g) was purified on a Silica gel column (3 kg,Eluent—Heptane/EtOAc from 4:1 to 1:1.5) to give 305 g (60% yield) of thedesired 1,6-di-O-benzoyl-2,5-anhydro-D-mannitol as white solid and 131 g(20%) of 1,3,6-tri-O-benzoyl-2,5-anhydro-D-mannitol.

Note 3:

Pivaloyl chloride may be used instead of benzoyl chloride afforded 50%of the desired 1,6-di-O-pivaloyl-2,5-anhydro-D-mannitol together with32% of 1,3,6-tri-O-pivaloyl-2,5-anhydro-D-mannitol.

Preparation of 2,5-Anhydro-3-O-(α-L-idopyranosyl)-D-mannitolhepta-O-sulfate Sodium Salt

Phenyl-6-O-acetyl-2,3,4-tri-O-benzoyl-1-thio-L-idopyranoside (125.4 g,0.196 mol) and 1,6-di-O-benzoyl-2,5-anhydro-D-mannitol (73.0 g, 0.196mol) were dissolved in dry CH₂Cl₂ (2000 ml) and the mixture was cooledto −30° C. under Argon. N-iodosuccinimide (56.2 g, 0.250 mol, 1.3 eq)was added in one portion. After 10 min of stirring, TMSOTf (5.7 ml, 29.5mmol, 0.15 eq) was added in one portion and the reaction was stirred foradditional 1 h (TLC monitoring, Heptane/EtOAc 1:1). The reaction wasquenched by addition of 10% aqueous NaHCO₃ (50 ml) followed by 10%aqueous Na₂SO₃ (1300 ml) and warmed to 25° C. Then, the phases wereseparated, the aqueous one was extracted with CH₂Cl₂ (1000 ml) and thecombined organics were washed Water (500 ml), dried over Na₂SO₄,filtered and evaporated under reduced pressure. The brown residue (250g) was dissolved in MeOH (900 ml) under reflux and then ˜100 ml of MeOHwas distilled off. The resulted mixture was cooled to 20° C., stirredfor 2 h and filtered. The cake was washed with cold MeOH (2×150 ml) andHeptane (150 ml) and dried under reduced pressure afforded 148.5 g (84%yield) of the desired product as off-white solid.

The partially protected disaccharide (70.0 g, 78.4 mmol) was suspendedin dry MeOH (800 ml) under Argon at 25° C. and Methanolic NaOMe (25% wt;18.0 ml, ˜78.5 mmol) was added in one portion. The reaction was stirredfor 12 h at 25° C. and reaction was quenched by addition of acetic acid(7 ml). All volatiles were evaporated, the residue was triturated withEtOAc (300 ml) at 60° C. for 1 h. The mixture was cooled to 0° C.,without stirring, stayed for 3 h and liquids were decanted. The solidresidue was crystallized from 240 ml of the mixture MeOH/2-Isopropanol(1:2), to give after filtration and drying 20.6 g (80% yield) of theunprotected disaccharide as white solid.

A suspension of the unprotected disaccharide (20.0 g, 61.3 mmol) in dryDMF (100 ml) was cooled to −20° C. under Argon and a solution of SO₃.DMF(˜48%, 143 g, 0.86 mol) in dry DMF (400 ml) was slowly added at a ratethat kept reaction temperature below −10° C. Thereafter the temperaturewas raised to 0° C., the reaction was stirred for 3 h and slowly poured(caution—gas evolution!) into the pre-cooled (0-4° C.) solution ofNaHCO₃ (145 g) in Water (1500 ml). The resulted mixture was stirred for20 min, neutralized with 10% aqueous H₂SO₄ and concentrated underreduced pressure. The solid residue was triturated with MeOH (700 ml),filtered and the solids were washed with MeOH (100 ml) and driedafforded 190 g of the desired polysulfate, contaminated with Sodiumsulfate. The crude was purified (by portions of 10 g) on a Sephadex G-25column (50-150 micron, 500 g) eluted with Water. All fractionscontaining the pure product were combined and evaporated under reducedpressure at 50° C. until constant weight to give 35.2 g (55% yield) ofthe desired heptasulfate as white powder.

The preferred product produced in this way was the hypersulfateddisaccharide having seven sulfate groups in the sodium salt form asshown below (SSS-02 or SSS-056-01 or compound 2)

or otherwise shown as:

Example 2-Pulmonary Evaluation of an Animal Model (Sheep)

To illustrate the effectiveness of the formulations according to theinvention to treat and alleviate allergen related diseases andconditions, including but not limited to the specific diseases andconditions recited herein, sheep were assessed in multiple experimentswhich compared various formulations containing no active ingredient toanimals which were provided formulations comprising a compound offormula I (SSS-02). To measure pulmonary airflow resistance, the sheepwere intubated with a cuffed nasotracheal tube and pulmonary airflowresistance (R_(L)) was measured by the esophageal balloon cathetertechnique. These methods are accepted and well known methods found inthe literature.

To assess airway responsiveness, cumulative dose response curves toinhaled carbachol were performed by measuring R_(L) before and afterinhalation of buffered saline and after each administration of 10breaths of increasing concentrations of carbachol (0.25, 0.5, 1.0, 2.0,and 4.0% wt/vol solution). Airway responsiveness was measured bydetermining the cumulative provocation dose (PD₄₀₀) of carbachol (inbreath units) that increased R_(L) to 400% above baseline. One breathunit was defined as one breath of 1% carbachol solution.

For airway studies, each animal's baseline airway responsiveness (PD₄₀₀)was determined and then, on different experimental days, the test sheepunderwent airway challenge with Ascaris suum antigen. R_(L) was measuredto establish baseline, then measured again immediately after antigenchallenge and hourly for an eight hour period and then a post challengePD₄₀₀ was measured 24 hours after antigen challenge. In each of theFigures presented herein, FIGS. 1A, 2A, 3A etc. present day two datameasured on an hourly basis for the eight hour period and containcontrol data (closed circles) and drug treatment data (open circles ortriangles (closed or open)). The drug treatment experiments wereconducted on the same animals used in the control studies but after aperiod of several weeks following the day 3 PD₄₀₀ measurements. FIGS.1B, 2B, 3B etc. contain the day one baseline PD₄₀₀ data and day threePD₄₀₀ data following antigen challenge in control or drug treatedanimals.

Data were expressed or may be expressed as (a) mean+/−SE % change ofR_(L) and (b) PD₄₀₀ in breath units. Data were also expressed as (c) %protection of Early Airway Response (EAR, for 0-4 hours) and Late AirwayResponse (LAR, for 4-8 hours), as estimated by area under the curve forEAR and LAR respectively. And (d)

${{AHR}\mspace{14mu} \% \mspace{14mu} {protection}} = {100 - {\frac{{{Baseline}\mspace{14mu} {PD}_{400}} - {{drug}_{antigen}{PD}_{400}}}{{{Baseline}\mspace{14mu} {PD}_{400}} - {{Control}_{antigen}{PD}_{400}}} \times 100}}$

Thus, the numbers in the Figures as presented were obtained by theformula: Baseline PD₄₀₀-drug_(antigen)PD₄₀₀ was x-y; BaselinePD₄₀₀−Control_(antigen)PD₄₀₀ was x−z. x−y/x−z×100=n. 100−n=X %protection in AHR.

In the studies presented in FIGS. 1A-9B, the data shows the % change inR_(L) and PD₄₀₀ in breath units for Control antigen response studies andfor Drug-Treated antigen response studies.

FIG. 1A shows a graph comparing the percentage change in pulmonaryairflow resistance (measured as cm H₂O/L/sec) (i.e., the R_(L))following the indicated time after antigen administration (time=0) ofsheep's responses (n=5) to exposure to antigen only (closed circles)(control) and antigen plus a liquid oral dosage of 0.5 mg/kg×4 days (QD)of the heptasulfated disaccharide (sodium salt) designated asSSS-056-01*. The last dose was administered ninety minutes beforeantigen challenge (i.e., −1.5 hr). Data are shown as antigen-inducedmean plus or minus SE % change in R_(L) in sheep (n=5) exposed toantigen first with no drug and then again several weeks later withantigen plus SSS-056-01.

FIG. 1B shows a bar graph illustrating the effect of pretreatment onairway hyperresponsiveness (AHR) in allergic sheep. Data are shown asmean plus or minus SE PD₄₀₀ (airway responsiveness) in breath units atbaseline, 24 hours post-antigen challenge in a group of sheep (n=5)exposed to antigen first with no drug and then again with antigenseveral weeks later following pretreatment (90 minutes beforehand) withan oral dose of SSS-056-01 (0.5 mg/kg×4 days (QD)) in liquid form. PD₄₀₀is defined as the provocating dose of carbochol in breath units whichcaused a 400% increase in R_(L). One breath unit is one breath of 1%solution of carbochol. PD₄₀₀ is an indicator of airway responsiveness.The 0.5 mg/kg oral dose (QD for 4 days) inhibited EAR by 77%, LAR by 95%and AHR by 100%.

FIG. 2A shows a graph comparing the percentage change in pulmonaryairflow resistance (i.e., the R_(L)) following the indicated time afterantigen administration (time=0) of sheep's responses (n=5) to exposureto antigen only (closed circles) (control) and antigen plus an iv dose(0.5 mgs/kg×4 days (QD) of the heptasulfated disaccharide designated asSSS-056-01 (open circles). The last does was administered 90 minutesbefore antigen challenge (i.e., 1.5 hrs) Data are shown asantigen-induced mean plus or minus SE % change in R_(L) in sheep (n=5)exposed to antigen first with no drug and then again several weeks laterwith antigen plus SSS-056-01. The results show that there is nodifference in bioavailability between the intravenous administered doseand the oral dose shown in FIG. 1A administered once a day.

FIG. 2B shows a bar graph illustrating the effect of pretreatment onairway hyperresponsiveness (AHR) in allergic sheep. Data are shown asmean plus or minus SE PD₄₀₀ (airway responsiveness) in breath units atbaseline, 24 hours post-antigen challenge in a group of sheep (n=5)exposed to antigen first with no drug and then again with antigenseveral weeks later following pretreatment (1.5 hours) with an i.v. Dose(0.5 mgs/kg×4 days (QD)) of SSS-056-01. The results again show thatthere is no difference between the i.v. route of administration(bioavailability) and the oral route shown in FIG. 1B. The 0.5 mg/kgi.v. dose (QD for 4 days) inhibited EAR by 77%, LAR by 92% and AHR by100%.

FIG. 3A shows a dose-response graph comparing the percentage change inpulmonary airflow resistance (measured as cm H₂O/L/sec) (i.e., theR_(L)) following the indicated time after antigen administration(time=0) of sheep's responses (n=5) to exposure to antigen only (closedcircles) (control) and antigen plus a liquid oral dosage at variousstrengths (0.25 mg/kg (open circles); 0.5 mg/kg (closed triangles) and 1mg/kg (open triangles)) of the heptasulfated disaccharide designatedSSS-056-01 (open circles). Data are shown as antigen-induced mean plusor minus SE % change in R_(L) in sheep (n=5) exposed to antigen firstwith no drug and then again several weeks later with antigen after beingpretreated with various oral doses of SSS-056-01 administered 90 minbefore antigen challenge.

FIG. 3B shows a bar graph illustrating the effect of pretreatment onairway hyperresponsiveness (AHR) in allergic sheep. Data are shown asmean plus or minus SE Paw) (airway responsiveness) in breath units atbaseline, 24 hours post-antigen challenge in a group of sheep (n=5)exposed to antigen first with no drug and then again with antigenseveral weeks later following pre-treatment with various oral doses ofSSS-056-01 (0.25, 0.5 and 1.0 mgs/kg) administered 90 min. before theantigen challenge. +P<0.05 vs. baseline; and *P<0.05 vs. antigencontrol. The data in FIGS. 3A and 3B demonstrate that a single oral doseof SSS-056-01 at 0.25 mg/kg was ineffective while 0.5 mg/kg and 1 mg/kginhibited LAR (71% and 77% inhibition) and AHR (100% inhibition) withoutan effect on EAR.

FIG. 4A shows a graph comparing the percentage change in pulmonaryairflow resistance (measured as cm H₂O/L/sec) (i.e., the R_(L))following the indicated time after antigen administration (time=0) ofsheep's responses (n=8) to exposure to antigen only (control, closedcircles), and antigen plus multiple (3× total; 1× every 12 hours) liquidoral doses of 0.25 mg/kg, 0.5 mg/kg and 1.0 mg/kg of the heptasulfateddisaccharide designated as SSS-056-01 (open circles, closed triangle,open triangles respectively; three weeks apart). Data are shown asantigen-induced mean plus or minus SE % change in R_(L) in sheep (n=8)exposed to antigen first with no drug and then again several weeks laterwith antigen after being pretreated before antigen exposure with 0.25mg/kg, 0.5 mg/kg and 1.0 mg/kg SSS-056-01 (1× each 12 hr period, 3 weeksapart). Antigen challenge was ninety minutes after the last dose.

FIG. 4B shows a bar graph illustrating the effect of pretreatment onairway hyperresponsiveness (AHR) in allergic sheep. Data are shown asmean plus or minus SE PD₄₀₀ (airway responsiveness) in breath units atbaseline, 24 hours post-antigen challenge in a group of sheep (n=8)exposed to antigen first with no drug and then again with antigenseveral weeks later following pre-treatment before exposure with aliquid oral dose of SSS-056-01 (0.25 mg/kg, 0.5 mg/kg and 1.0 mgs/kg; 3weeks apart) administered 1×3 each 12 hours. Antigen challenge wasninety minutes after the last mg/kg dose. The results show that theeffect of multi-dose oral SSS-056-01 is cumulative. While 0.25 mg/kg×3doses is ineffective; 0.5 mgs/kg×3 and 1 mg/kg×3 inhibited EAR (30% and54% inhibition), LAR (85% and 87% inhibition) and AHR (100% inhibition).1 mg/kg caused significantly greater inhibition of EAR than 0.5 mg/kgwhile the effect on LAR and AHR were comparable.

FIG. 5A shows the area under the curve for early phase(AUC-EAR_(0-4 hr)) from the data obtained in FIG. 4A.

FIG. 5B shows the area under the curve for the late phase(AUC-LAR_(4-8 hr)) from the data obtain in FIG. 4A.

FIG. 6A shows a graph comparing the percentage change in pulmonaryairflow resistance (measured as cm H₂O/L/sec) (i.e., the R_(L))following the indicated time after antigen administration (time=0) ofsheep's responses (n=5) to exposure to antigen only (closed circles)(control) and antigen plus a liquid oral dosage of 0.5 mg/kgadministered twice a day (BID) for a total of seven doses of theheptasulfated disaccharide designated as SSS-056-01 (open circles). Datashown are antigen-induced mean plus or minus SE % change in R_(L) insheep (n=5) exposed to antigen first with no drug and then again severalweeks later with antigen after being pretreated before antigen exposurewith the seven doses. The last dose was administered 90 minutes beforethe antigen challenge.

FIG. 6B shows a bar graph illustrating the effect of pretreatment onairway hyperresponsiveness (AHR) in allergic sheep. Data are shown asmean plus or minus SE PD₄₀₀ (airway responsiveness) in breath units atbaseline, 24 hours post-antigen challenge in a group of sheep (n=5)exposed to antigen first with no drug and then again with antigenseveral weeks later following pretreatment with seven total dosesadministered every 12 hours before exposure with an oral dose ofSSS-056-01 (0.5 mg/kg). Antigen exposure occurred ninety minutes afterthe last 0.5 mg/kg treatment. The data in FIGS. 6A and 6B show that theeffect of multidose BID dosing of oral SSS-056-01 is cumulative. 0.5mg/kg oral doses (BID×7 doses) inhibited EAR by 76%, LAR by 96% and AHRby 100%. This is significantly better than 0.5 mg/kg×3 doses. BID dosing(×7) is comparable to QD (×4) dosing.

FIG. 7A shows the effect of multi-dose oral SSS-056 on antigen-inducedEAR, LAR and AHR in sheep (QD dosing). Oral SSS-056-01 (0.5 mg/kg) wasadministered once daily in the morning×4 days, and antigen challenge wasperformed 90 minutes after the last dose (n=5). EAR and LAR are shown asantigen-induced % change in R_(L)±SE, without (control, closed circles)and after treatment with oral SSS-056-01 (open circles). The resultsalso show that the effect of multi-dose oral SSS-056-01, BID versus QDdosing is comparable.

FIG. 7B shows post-antigen AHR shown as mean±SE PD₄₀₀ for the baselineand 24 hours post-antigen without (control) and after treatment withSSS-056-01. +P<0.05 vs. Baseline; *P<0.05 vs. antigen control.

FIG. 8A shows the effect of single dose “inhaled” SSS-056 onantigen-induced EAR, LAR and AHR in sheep (n=5). Inhaled SSS-056-01 inbacteriostatic injection water was administered 30 minutes beforeantigen challenge. EAR and LAR are shown as antigen-induced % change inR_(L)±SE without (control) and after treatment with various doses ofSSS-056-01.

FIG. 8B shows post antigen AHR shown as mean+/−SE PD₄₀₀ for the baselineand 24 hour post-antigen, with (control) and after treatment with 5 mgand 10 mg inhaled SSS-056-01. This shows that 10 mg single dose ofinhaled SSS-056-01 inhibits LAR (75% inhibition) and AHR (100%inhibition) without an effect on EAR; while the 5 mg dose wasineffective.

FIG. 9A shows multi-dose inhaled SSS-056-01 (10 mg×3) had no cumulativeeffect on EAR (n=6), but did have an effect on LAR. The data wascomparable to a single dose, as shown in FIG. 8.

FIG. 9B shows post antigen AHR shown as mean SE PD400 for the baselineand 24 hour post-antigen, with (control) and after treatment with 10mg×3 inhaled SSS-056-01. This shows that multiple dose (10 mg×3) ofinhaled SSS-056-01 inhibits LAR (75% inhibition) and AHR (100%inhibition) without an effect on EAR

FIG. 10 shows the proton NMR of SSS-056-01(2,5-anhydro-3-O-(α-L-idopyranose)-D-mannitol-hepta-O-sulfate-heptasodium salt).

*This compound is also described herein as SSS-02.

While the claimed invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade to the claimed invention without departing from the spirit andscope thereof. Thus, for example, those skilled in the art willrecognize or be able to ascertain using no more than routineexperimentation, numerous embodiments of the claimed invention which maynot have been expressly described. Such embodiments are within the scopeof the invention.

1. A dosage form comprising a substantially pure form of a compound offormula I or pharmaceutically acceptable salts thereof

wherein R₁-R₇ are independently selected from the group consisting ofSO₃H or PO₄H and a pharmaceutically acceptable excipient wherein theamount of a compound of formula I in said dosage form ranges from 0.3 to10 mgs/kg.
 2. The formulation according to claim 1 wherein R₁-R₇ isselected from SO₃H.
 3. The formulation according to claim 1 wherein thesalt is a sodium salt.
 4. The formulation according to claim 1 whereinsaid formulation is oral.
 5. The formulation according to claim 1 whichis i.v.
 6. The formulation according to claim 1 in inhalable form.
 7. Amethod of treating or alleviating an inflammatory condition in a mammalin need of treatment thereof comprising administration of (i) apharmaceutically effective amount of a formulation comprising a compoundof formula I

and pharmaceutically acceptable salts thereof wherein R₁-R₇ areindependently selected from SO₃H or PO₄H and (ii) a pharmaceuticallyacceptable excipient.
 8. The method according to claim 7 wherein theinflammatory condition is selected from pulmonary inflammation such asasthma and/or asthma related pathologies; pneumonia, tuberculosis,rheumatoid arthritis, allergic reactions which impact the pulmonarysystem, early and late phase responses in asthma and asthma relatedpathologies, diseases of the small and large airways of the lung,bronchospasm, inflammation, increased mucus production, conditions whichinvolve vasodilation, plasma exudation, recruitment of inflammatorycells such as neutrophils, monocytes, macrophages, lymphocytes andeosinophils and/or release of inflammatory mediators by resident tissuecells (mast cells); conditions or symptoms which are caused byallergens, secondary responses to infections, industrial or occupationalexposures, ingestion of certain chemicals or foods, drugs, exercise orvasculitis; conditions or symptoms which involve acute airwayinflammation, prolonged airway hyperreactivity, increases in bronchialhyperreactivity, asthmatic exacerbations, hyperresponsiveness;conditions or symptoms which involve the release of inflammatorymediators such as 15-HETE, leukotriene C4, PAF, cationic proteins oreosinophil peroxidases; conditions or symptoms which relate tocutaneous, nasal, ocular or systemic manifestations of late phaseallergic responses; clinical diseases of the skin, lung, nose, eye orthroat or other organs and which involve allergic mechanisms having anhistologic inflammatory component upon antigen challenge; allergicrhinitis, respiratory diseases characterized by seasonal or perennialsneezing; rhinorrhea, conjunctivitis, pharyngitis, intrinsic orextrinsic asthma bronchiale, any inflammatory lung disease, acutechronic bronchitis, pulmonary inflammatory reactions secondary to acutechronic bronchitis, chronic obstructive lung disease (COPD), cysticfibrosis, ARDS, acute lung injury, pulmonary fibrosis, Goodpasture'ssyndrome, any pulmonary condition in which white blood cells play a roleincluding but not limited to idiopathic pulmonary fibrosis and any otherautoimmune lung disease; ear, nose and throat disorders such as acuteexternal otitis, furunculosis and otomycosis of the external ear;respiratory diseases such as traumatic and infectious myringitis, acuteeustachian salpingitis, acute serous otitis media, acute and chronicsinitis; extrapulmonary conditions selected from any late-phasereactions and inflammatory response such as allergic rhinitis; allergicdermatitis; allergic conjunctivitis; extrapulmonary diseases whereinflammation occurs and/or an inflammatory response plays a major roleincluding inflammatory bowel disease; central nervous system disorders;neuroinflammation; Alzheimer's, the inhibition of β-amyloid aggregationin Alzheimer's disease, rheumatoid arthritis and other collagen vasculardiseases; glomerulonephritis; inflammatory skin diseases and sarcoidosisand cardiovascular inflammation.
 9. The method according to claim 8wherein the mammal in need of treatment thereof is human.
 10. A processfor producing a compound of formula I comprising the steps of (1)reacting a thioglycoside of formula Ia with a mannitol of formula Ib toform a compound of formula Ic; (2) reacting the compound of formula Icwith a sulfating reagent to form a compound of formula I and wherein thethioglycoside Ia was produced using1,6-anhydro-3-O-benzyl-βL-idopyranose as an intermediate.
 11. Theprocess according to claim 10 wherein the compound of formula Ia isproduced by a process which comprises (a) reacting1,6-anhydro-3-O-benzyl-β-L-idopyranose (3)

with a reducing agent and benzoyl chloride to form compound 4:

(b) reacting compound 4 with an acetylating agent to form compound 5:

(c) thiolating compound 5 with a thiolating reagent to form a compoundof formula Ia.
 12. The process according to claim 1I wherein pivaolylchloride is used as a reagent in place of beyzoyl chloride to formcompound 4′

and compound 4′ is acetylated to form compound 5′

which is further reacted with a thiolating reagent to form the pivolatedversion of a crystalline form of compound Ia.
 13. The process accordingto claim 11 wherein the thiolating reagent is selected from thiophenolor thiocresol.
 14. A process for producing a substantially pure compoundof formula I comprising the steps of (a) reacting a compound of formulaIa with a compound of formula Ib wherein the protecting group (Pr) incompounds Ia and Ib are identical.
 15. A substantially pure crystallineform of 2,5-anhydro-3-O-(α-L-idopyranosyl)-D-mannitol hepta-O-sulfatesalt wherein the salt is substantially free of impurities.
 16. Thesubstantially pure crystalline form according to claim 15 wherein thesalt is a sodium salt and the impurities are acetate salts.
 17. Thecrystalline form according to claim 16 having less than 1.4 wt % ofsodium acetate as an impurity.
 18. The form according to claim 17 havingless than 1.0 wt % of sodium acetate.
 19. The form according to claim 17having less than 0.5 wt % of sodium acetate.